JPH0947503A - Base material for cultivated skin, cultivated skin, and method of making it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To smoothly supply enough nutriments to cultivated epidermis cells and/or fibroblasts without any cells falling/removing when a collagen base material is seeded, and to smoothly discharge exuding liquid from the surface of a wound to which the cultivated skin is applied. SOLUTION: The cultivated skin consists of a collagen base material which has through notches and fibroblasts/epidermis cells seeded/cultivated at least on one side of the collagen base material. If force is added in the directions to widen the notches at the time of application of the cultivated cells, through holes are formed at this time.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a substrate for cultured skin, cultured skin and a method for producing the same. More specifically, the present invention relates to a substrate for cultured skin, a cultured skin, and a method for producing the same for use in skin defect wounds such as skin diseases such as burns, wounds, pressure ulcers or skin ulcers, for early reconstruction or treatment of defective tissues. .

[0002]

2. Description of the Related Art Conventionally, as a treatment for a skin defect wound reaching a shallow portion of the dermis such as superficial degree II burns, a wound dressing or an ointment-applied gauze has been generally used. Is. However, reconstruction of skin tissue by self-proliferation of epidermal cells cannot be expected in the case of extensive skin defect reaching deep part of dermis such as deep-seated II burn, III burn or pressure ulcer more than II. Therefore, a treatment has been performed in which necrotic tissue or poor granulation tissue is first removed, and then allogeneic skin or wound dressing is applied to reconstruct good granulation tissue, and then skin is reconstructed by autologous split-layer skin grafting. It was However, in the case of autologous split-layer skin grafting, although skin is collected from a healthy part of the self, there is a problem with dressing that scars remain on the skin-collected part. Also, when the affected area is extremely wide, it is difficult to carry out autologous split-layer skin grafting.

In order to solve this problem, a technique has been developed in which epidermal cells or fibroblasts are collected from a small amount of skin and mass-cultured in a culture flask, and various cultured skins using these cultured cells have been developed. Has been developed.

Howard Green,
James Reinwald
The cultured epidermal sheet developed by them is one that obtains an epidermal sheet by collecting stamp-sized skin and culturing the epidermal cells in a large amount in a culture flask, but by enzymatic treatment when peeling the sheet from the culture flask. It has been pointed out that the engraftment rate is low in autologous transplantation because the most important cells, basal cells, are damaged (see Norimitsu Kuroyanagi, Vol. 9, Biomaterials, February 1991).

Also, Eugene Bell
Et al. (Science), Volume 211, 105
2-1054, March 1981), a large amount of epidermal cells and fibroblasts separated and collected from the skin are cultured in a culture flask, and the epidermal cells are layered on a collagen gel incorporating the fibroblasts. The developed cultured skin was developed.

Furthermore, Stephen Voice (S. Bo
yce) et al. (SURGERY, 103
Vol. 421-431, April 1988), a collagen sponge obtained by adding a small amount of chondroitin-6-sulfate obtained by culturing a large amount of epidermal cells collected from the skin in a culture flask. We are developing a cultured skin that is seeded on top and stratified.

Further, cultured skin based on atelocollagen is described in JP-A-62-246371 and JP-A-4-332561. Japanese Unexamined Patent Publication No. 62-246371 describes cultured skin using an atelocollagen sheet as a base material, but since the sheet has no through holes, nutrients indispensable for cell growth and the like brought from the transplant bed. Since it is difficult to permeate through the sheet to be supplied to the epidermal cells on the sheet, there is a problem that the epidermal engraftment rate is low in autologous transplantation.

[0008] Further, even when a cultured skin incorporating only fibroblasts is produced, it is difficult to supply nutrients from the applied wound surface to the whole cultured skin substrate, so that the proliferation and metabolism of fibroblasts can be suppressed. Inactivity and the release of physiologically active substances are not smooth, which may hinder the reconstruction of the skin or the formation of benign granulation tissue.

Further, since there is no through-hole, there is a problem that the exudate is excessively retained in the affected area to which the cultured skin is applied, and this discharge is not smoothed and becomes a hotbed of infection.

Japanese Unexamined Patent Publication (Kokai) No. 4-332561 discloses an atelocollagen sponge having through holes for solving the above-mentioned problems. However, when the through holes are provided, cells are seeded when seeded. In order to prevent this, it is essential to coat the sponge with atelocollagen gel or the like before seeding the cells in order to prevent this, and such a method not only complicates the process but also costs more for cultured skin. There is a problem that becomes. Therefore, it has been desired to develop a cheaper and simpler alternative or method.

[0011]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention Conventionally, there have been problems in cultured skin using collagen as a base material. After the application to the skin-deficient tissue, the cultured skin substrate serves as a barrier, and the nutrient supply from the applied wound surface to the entire cultured skin cannot be carried out smoothly. 2. Excessive accumulation of exudate in the cultured skin application site after application to skin-deficient tissue. When a through hole is provided in the base material in order to prevent the above 1 and 2, the cells fall off during cell seeding, and 4. If the through holes of the base material are coated with a biofibrous polymer material such as collagen gel in order to prevent the above item 3, the cost is increased.

The present invention has been made to solve the problems 1 to 4 described above, and is a culture for early reconstruction of a defective tissue by using it for a skin defect wound such as a burn, a wound, a pressure ulcer or a skin ulcer. Intended to provide skin. More specifically, it is intended to provide a cultured skin which has a function of smoothly supplying nutrients from the applied wound surface to cells of the whole cultured skin and discharging the exudate excessively retained on the applied wound surface, and which can be produced at low cost. With the goal.

[0013]

[Means for Solving the Problems] The above-mentioned object is to prevent the cells from falling and escaping when seeding cells derived from the skin on the substrate by providing a notch penetrating the collagen substrate for cultured skin. It does not require the intended treatment and creates a through hole by applying a force to open the incision when applying the cultured skin, providing nutrients to the cells seeded and cultured on the base material, and excessive application to the wound surface. The purpose of the present invention is to provide cultured skin in which the exudate accumulated in the skin can be discharged smoothly.

Therefore, the present invention relates to a substrate for cultured skin, which comprises a collagen sponge or a collagen sheet having a through cut.

The incision may be linear and may be provided along one direction, and may be provided at least partially in a 2.5 cm × 2.5 cm square in the substrate for cultured skin. In addition, a square having a size of 1 cm × 1 cm may not include a cut having a total length of more than 4 cm.

[0016] The present invention also relates to a cultured skin comprising a collagen sponge or a collagen sheet provided with a through cut and a cultured skin comprising cells derived from seed-cultured skin.

Further, the present invention provides a cultured skin comprising a collagen skin sponge or a collagen sheet provided with a through cut, and a skin-derived fibroblast derived from the skin seed-cultured on at least one surface of the substrate. Yes,
Cultured skin that can generate a through hole by applying a force to open the cut when applying the cultured skin, a cultured skin substrate made of a collagen sponge or a collagen sheet provided with a through cut and one side of the substrate It is a cultured skin consisting of epidermal cells that have been seeded and cultured, and a cultured skin that can generate a through hole by applying a force to open the cut when the cultured skin is applied, and a collagen sponge or a collagen sheet provided with the through cut. A cultured skin comprising a base material for cultured skin and a skin-derived fibroblast seeded and cultured on at least one surface of the base material, and epidermal cells seeded and cultured on one surface of the base material. The present invention relates to a cultured skin that can generate a through hole by applying force so as to open an incision.

The collagen may be atelocollagen, and the cuts may be evenly provided.

The cut may be a straight line provided along one direction, and a through hole may be formed by applying a force to open the cut, or
At least partially provided in a 2.5 cm × 2.5 cm square in the culture skin substrate, and 1 cm
A square of × 1 cm may not include a cut having a total length of more than 4 cm, or may be a slit. More preferably, a force is applied so that the notch opens in a direction that is not parallel to the notch direction.

Further, the present invention comprises: a) a step of providing a notch penetrating a cultured skin substrate comprising a collagen sponge or a collagen sheet, and b) a fiber derived from skin on at least one surface of the substrate with the notch closed. A method for producing cultured skin, which comprises a step of inoculating and culturing blast cells, and a) a step of providing a cut-through in a substrate for cultured skin made of a collagen sponge or a collagen sheet, and b) the base material with the cut being closed. A method for producing a cultured skin, comprising the step of seeding and culturing epidermal cells on one side of a), and a) a step of providing a cut-through in a culture-skin substrate comprising a collagen sponge or a collagen sheet, and b) in a state where the cut is closed. Skin-derived fibroblasts are seeded and cultured on at least one surface of the base material, and epidermal cells are seeded on one surface of the base material. A process for producing a cultured skin which comprises the step of culturing.

The collagen may be atelocollagen, and the incisions may be evenly provided.

The notch may be a straight line provided along one direction, and a through hole may be formed by applying a force to open the notch.
At least partially provided in a 2.5 cm × 2.5 cm square in the culture skin substrate, and 1 cm
A square of × 1 cm may not include a cut having a total length of more than 4 cm, or may be a slit. More preferably, a force is applied so that the notch opens in a direction that is not parallel to the notch direction.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, "closed state"
The term "is a state in which no force is applied to open the cut in the cultured skin substrate.

The substrate for cultured skin of the present invention includes collagen, gelatin, chitin, chitosan, polyglycolic acid,
Mucopolysaccharides such as chondroitin sulfate or hyaluronic acid can be used, and the collagen is preferably biocompatible such as atelocollagen.

The collagen sponge used in the present invention is prepared by homogenizing an acidified collagen solution with a homogenizer, pouring it into a container such as polystyrene containing a sufficient amount of air bubbles, and allowing it to stand in an ammonia gas atmosphere. After gelling, wash with water,
It can be produced by performing freeze-vacuum drying, and then introducing intermolecular crosslinking by ultraviolet irradiation or a crosslinking agent.

In the present invention, the collagen solution is prepared from collagen obtained from bovine dermis or the like, the pH is adjusted to preferably 2 to 4, and the concentration is 0.2 to 3 w / v%.
It is preferably obtained by setting the content to 0.5 to 2 w / v%. The gelation is carried out in a gas atmosphere such as ammonia for several minutes to 2 hours, if necessary, after which it is washed with water and freeze-dried under vacuum.

The dominant wavelength of ultraviolet rays (UV) used for crosslinking is 2
50 to 270 nm is preferable, and the amount of ultraviolet rays is 500
~ 12000 mWsec / cm ² , preferably 1000
Irradiation with a dose of up to 5000 mWsec / cm ² is recommended. Examples of the cross-linking agent used in the present invention include:
Glutaraldehyde, ethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether,
Examples thereof include glycerol polyglycidyl ether and hexamethylene diisocyanate. The size of the collagen sponge is appropriately selected according to the size of the affected area. The thickness and shape of the collagen sponge varies depending on the condition of the affected area to which the collagen sponge is applied.
The shape is 40 mm, and the shape may be a thin plate shape, a plate shape, a rod shape, a spindle shape, or a biconvex lens shape.

The collagen sheet used in the present invention is
The collagen solution can be produced by placing the collagen solution in the same container as used for the production of the sponge, air-drying in a drier to form a sheet, and then introducing ultraviolet rays or introducing an intermolecular crosslink with a crosslinking agent.

The collagen solution used for preparing the collagen sheet is prepared from collagen obtained from bovine dermis or the like, the pH is adjusted to preferably 2 to 4, and the concentration is 0.2 to 3 w / v%, preferably Is 0.5 to 2 w / v%
Is obtained.

The dominant wavelength of ultraviolet rays (UV) used for crosslinking is 2
50 to 270 nm is preferable, and the amount of ultraviolet rays is 500
~ 12000 mWsec / cm ² , preferably 1000
Irradiation with a dose of up to 5000 mWsec / cm ² is recommended. Examples of the cross-linking agent used in the present invention include:
Glutaraldehyde, ethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether,
Examples thereof include glycerol polyglycidyl ether and hexamethylene diisocyanate. The size and thickness of the collagen sheet are appropriately selected according to the condition of the affected area.

The penetrating incision provided in the substrate for cultured skin of the present invention means a line having a very thin blade and a sharp cutting edge, such as a cutting tool such as a safety razor or a knife. When the cutting means is removed, there is almost no gap where cells fall and escape. Therefore, when seeding cells, it is extremely unlikely that the cells will fall and escape through the cuts provided, and when applying the cultured skin, widen the cuts by applying a force to extend the base material. Through holes can be formed through which nutrients and exudates from the applied wound surface can pass.

The blade used for providing the above-mentioned notch is thinner the thinner the blade edge is, and the thicker the blade is 0.2 m.
It does not exceed m, and is preferably within 0.1 mm.
The number of cuts is not particularly limited, but is 2 to 20 m.
m, preferably 5-10 mm long incision in the longitudinal direction with respect to the incision 2-30 mm, preferably 5-15 mm
3 to 20 mm in the lateral direction at intervals, preferably 5 to 15 m
The cuts between adjacent rows at m intervals are staggered 1
It is preferable to provide the cuts adjacent to each other row (see FIG. 2B), but the cuts of adjacent rows may be arranged adjacent to each other (FIGS. 2A and 2C). reference). Further, it is preferable that the cuts are evenly provided on the substrate for cultured skin, but the length and density of the cuts squeezed on the same substrate may be different.

The shape of the cut is not only linear, but also semi-arc, corrugated, keyed, radial linear, concentric circular,
It may be a concentric oval shape, a cup shape, or an amorphous shape, but a shape that easily opens when a force in a certain direction is applied is preferable. All the cuts may have the same shape or different, and the arrangement may be not only parallel but also random. As examples of these, the shapes are shown in FIGS. 1 to 9, but the present invention is not limited thereto.

The cultured skin substrate of the present invention comprises mammalian skin-derived epidermal cells and / or at least one surface thereof.
Alternatively, fibroblasts are seeded and grown in culture. The term "epidermal cells" refers to keratinocytes including basal cells and other cells usually present in the epidermal cell layer. Among them, keratinocytes are cultured and proliferated. The fibroblast is a main cell in the dermis, and refers to a cell that produces connective tissue components such as collagen and binds to these components to form connective tissue.

The cultured skin of the present invention can be prepared in the following three types depending on the cells to be inoculated: Inoculation culture of only skin-derived fibroblasts on at least one surface of the collagen substrate of the present invention. Cultured skin (composite culture dermis), cultured skin in which epidermal cells were seeded and cultured on one side of the collagen substrate of the present invention (composite culture epidermis), and skin-derived fibroblasts were seeded on at least one side of the collagen substrate of the present invention Cultured skin obtained by culturing and seeding culture of epidermal cells on one surface of the substrate (composite culture skin). In this case, it is more preferable to first carry out the step of seeding and culturing the fibroblasts, because the epidermal cells can be prevented from falling off.

The above-mentioned three types of cultured skin are
The effect can be more effectively exhibited by properly using them according to the depth and the symptoms of the patient. In addition, these cultured skins can be properly used at home and at other homes.

The epidermal cells are prepared by the following procedure.
The skin (part of the epidermis and dermis, or the entire skin layer) collected in a clean environment is disinfected and immersed in a physiological saline solution containing an antibiotic or a buffer solution such as Hank's solution. The dispase concentration of this skin is 1000
Dulbecco's modified Eagle's minimum essential medium (DMEM) prepared to U / mL (hereinafter referred to as "dispase solution")
After soaking in, separate into epidermis and dermis. The obtained epidermis was prepared in a Hanks solution (hereinafter, referred to as a "trypsin solution") in which the trypsin concentration was 0.25 w / v% and the sodium ethylenediaminetetraacetate (EDTA) concentration was 0.5 mM.
After soaking at 37 ° C. for about 15 minutes, DMEM containing 10 v / v% fetal calf serum (FCS) (hereinafter, this medium is referred to as “D
MEM + 10% FCS "), etc.
Disperse the cells by shaking, about 400 × g,
It can be obtained by centrifuging for 5 minutes and collecting. The obtained epidermal cells are, for example, green (G
reen) medium, NCTC168 medium, MCDB153
An epidermal cell suspension is prepared by adding a medium, particularly preferably Green medium.

The green medium is DMEM and ham (H
am's) F-12 was mixed in a ratio of 3: 1, and hydrocortisone (0.4 μg / mL) and insulin (5 μg / m)
L), transferrin (5 μg / mL), triiodothyronine (0.0013 μg / mL), cholera toxin (0.01 μg / mL), adenine (24.3 μg / m).
L), epidermal growth factor (0.01 μg / mL) and antibiotics, and an epidermal growth medium containing 10 v / v% FCS (Cell, 40, 677).
Pp. 683, March 1985 issue).

In order to grow the epidermal cells with high efficiency,
For example, it is a mouse-derived fibroblast whose proliferative capacity has been lost by treatment with mitomaincin or irradiation.
It is preferable to carry out the culture in a culture flask to which T3 cells and the like are attached as supporting cells.

Specifically, after culturing the 3T3 cells, the medium is removed and rinsed with Hank's solution to remove it. Then DME containing mitomycin C 4 μg / mL
Add the M solution so that the whole cells are sufficiently immersed (in a 75 cm ² culture flask, about ^{2 to 4} mL is preferable) and add 37 ° C.
After leaving it to stand for about 2 hours, it is washed with a buffer solution to remove mitomycin C. In this way, the 3T3 cells are alive, and only the proliferative ability is lost. The obtained 3T3 cells having no proliferative ability are collected, suspended in the green medium, and then 1 × 10 ^{3 to} 5 × 10 ⁴ cells / cm ² , preferably 5 × 10 ^{3 to} 3 × 10 ⁴ cells / It is prepared to have a density of cm ² , and then seeded and adhered to a culture flask. 3T3 cells were seeded on the culture flask with 3T3 cells, and then 5 × 10 ^{3 to} 5 × 10 ⁵ cells / c were seeded.
m ² , preferably 1 × 10 ⁴ to 2 × 10 ⁵ cells / c
Seed at a cell density of m ² and culture at 37 ° C. in a 5% CO ₂ incubator.

The 3T3 cells are detached from the bottom surface of the culture flask during the process of forming colonies of the epidermal cells during the continuation of the culture, and float up in the medium, and are removed when the medium is replaced. Is hardly included in.

The epidermal cells grown in culture were peeled off by adding a dispase solution and leaving it at 37 ° C. for about 2 hours.
Further, it is dispersed with a trypsin solution and centrifuged to collect epidermal cells, and a green medium is added to obtain a cell suspension. The epidermal cells are subcultured as necessary to obtain a large amount of epidermal cells. Specifically, the required number of the supporting cells are prepared, and the epidermal cells cultured and grown as described above are collected and seeded and cultured on the supporting cells. At this time, subculture is performed so that epidermal cells are not keratinized. The obtained epidermal cells are, for example, 5 × 10 ⁴ to 5 × 10 ⁴ on a collagen sponge substrate having a notch and having a thickness of about 6 × 9.5 cm and a thickness of 1 mm.
The cell density is adjusted to 2 × 10 ⁵ cells / cm ² and seeded.

FCS was added to the entire substrate prior to cell seeding,
Let stand at 37 ℃ for about 20 hours, then add extra FCS
By removing the cells, the adhesion of the cells is improved. After the epidermal cells have adhered to the substrate, a green medium is added and the cells are cultured in a 5% CO ₂ incubator at 37 ° C. for 7 to 21 days while changing the medium every 3 days. In this way, a composite culture epidermis is obtained.

For the fibroblasts, the collected skin is separated into epidermis and dermis in the same manner as described above, the obtained dermis is crushed with scissors, a homogenizer, etc., and collagenase is dissolved in DMEM to give 0.5 w / The solution was added to a solution prepared to have a concentration of v% (hereinafter, referred to as "collagenase solution"), and allowed to stand at about 37 ° C for about 3 hours, and then shaken to dissolve the connective tissue. Collect by centrifugation at 1,000 xg, preferably about 600 xg to about 800 xg. The obtained fibroblasts are cultured in DMEM + 10% FCS or the like as a medium at 37 ° C. in a 5% CO ₂ incubator until subconfluence, and subcultured as necessary to obtain many fibroblasts. .

When the obtained fibroblasts are seeded, for example, on a collagen sponge substrate having a cut size of about 6 × 9.5 cm and a thickness of 1 mm, first, before seeding, in the above-mentioned preparation of the composite culture epidermis. Similarly, FCS is included in the base material. The cultured fibroblasts were detached from the culture flask using trypsin solution, collected by centrifugation, and
A suspension is prepared by adding MEM + 10% FCS. This fibroblast suspension was mixed with 5 × 10 ^{3 to} 5 × 10 ⁵ cells.
/ Cm ² , preferably 5 × 10 ⁴ to 2 × 10 ⁵ cells
Seed the collagen substrate at a cell density of / cm ² . After the cells have adhered to the substrate, DMEM + 10% FCS is added, and the cells are cultured at 37 ° C. in a 5% CO ₂ incubator for 1 to 14 days while changing the medium every 3 days. In this way, a composite cultured dermis can be obtained. The composite cultured dermis is obtained by culturing and proliferating fibroblasts on at least one side of a substrate.

A composite cultured skin having both epidermal cells and dermal cells together is prepared as follows. Fibroblasts are seeded in the above-described step of producing the composite cultured dermis, the cells are favorably adhered to the substrate, the substrate is turned over, and the step of producing the composite cultured epidermis is performed to obtain a composite cultured skin.

The cultured skin thus obtained is preferably washed with DMEM and replaced with a similar medium without FCS before being applied to the wound surface.

With cultured skin using a substrate having no through-holes, it was difficult to supply nutrient components to the entire cultured skin, which sometimes inhibited the growth and metabolism of cultured cells. In addition, exudates, etc. were applied to the wound surface to which it was applied. May excessively accumulate and become a hotbed of infection, whereas in the cultured skin using the base material of the present invention, as described above, force is applied to open the squeezed incision before application to the wound surface. In addition, by providing the through holes, the nutrient components are easily supplied to the entire cultured skin, the growth and metabolism of the cultured cells are facilitated, and the exudate on the applied wound surface is satisfactorily discharged.

When autologous transplantation of the combined culture epidermis or the combined culture skin is effective in increasing the survival rate.

In order to make the above-mentioned cells adhere well to the substrate, even better cultured skin can be obtained by coating the substrate of the present invention with fibronectin, laminin or the like as a cell adhesion factor.

[0051]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Atelocollagen Sponge with Notches A homogenizer (manufactured by Nippon Seiki Seisakusho Co., Ltd.) was prepared by using an aqueous solution (adjusted to pH 4 with hydrochloric acid) of atelocollagen (manufactured by Koken Co., Ltd.) prepared to 1 w / v%. ) Was used to stir for 1 minute to introduce air bubbles, and 50 ml was poured into a plastic case (10 × 18 cm, height 2.8 cm) to cause gelation in an ammonia gas atmosphere. This was thoroughly washed in running water and freeze-dried under vacuum to obtain an atelocollagen sponge having a thickness of about 1 mm. Then
One side of this sponge was irradiated with UV at 1.5 mW / cm ^{2 for} 30 minutes each on the front and back sides.

Using a stainless steel knife (made by Feather Safety Razor Co., Ltd.) on the obtained atelocollagen sponge, cuts having a length of 5 mm were arranged at intervals of 5 mm parallel to the long side and 5 mm at the short side. The cuts were staggered, and the cuts were pierced in every other row so that the cuts were adjacent to each other, and ethylene oxide gas sterilization was performed to obtain a cut atelocollagen sponge for cultured skin (Fig. 1).
(A)).

Example 2 Preparation of incised composite culture skin Human skin pieces collected in a clean environment (about 2 x 2c)
m, thickness about 0.5 mm) was dipped in an isodine solution (manufactured by Meiji Seika Co., Ltd.), washed with Hanks' solution, then streptomycin (1000 μg / mL), penicillin (1000 U / mL) and amphotericin B (2.5).
(g / mL) was mixed with Hanks' solution for 30 minutes at room temperature to sterilize.

Next, 10 ml of a dispase solution (Dispase manufactured by Godo Shusei Co., Ltd., DMEM manufactured by Lifetech Technologies) was immersed at 4 ° C. for about 20 hours and separated into epidermis and dermis using tweezers. The epidermis thus obtained was mixed with 10 ml of a trypsin solution (trypsin manufactured by Wako Pure Chemical Industries, Ltd., EDTA manufactured by Dojindo Co., Ltd.).
DMEM + 10% FC after treatment at 37 ℃ for 5 minutes
The cells are dispersed in the medium by transferring to S and shaken, collected by sedimentation by centrifugation at about 400 × g for 5 minutes, and suspended in a green medium having the composition described herein. did.

The following feeder cells were used to grow epidermal cells with high efficiency. It is a mouse-derived fibroblast 3
T3 cells were cultured at 37 ° C. in a 5% CO ₂ incubator until subconfluent in a culture flask containing DMEM + 10% FCS. After that, the medium is removed and rinsed with Hank's solution, and DMEM is added thereto so that the final concentration of mitomycin C is 0.0004%, and physiological saline solution containing mitomycin C (manufactured by Wako Pure Chemical Industries, Ltd.) is added. (0.1 mg / mL) was added. Then, this culture flask was allowed to stand at 37 ° C. for 2 hours, then washed with Hank's solution to remove mitomycin C, 3T3 cells in which the proliferation ability had disappeared were collected, and further suspended in green medium, and after counting, 2 × 10 ⁴ The cells were prepared to have a density of cells / cm ² and seeded in a new culture flask.

The above-prepared epidermal cells were seeded in a culture flask to which 3T3 cells thus prepared adhered, and cultured and grown at 37 ° C. in a 5% CO ₂ incubator.

On the other hand, the separated dermis was crushed with scissors until it became a paste, and a collagenase solution (collagenase manufactured by Wako Pure Chemical Industries, Ltd., DMEM manufactured by Lifetech Technologies, Inc.) was heated to 37 ° C. for about 6 hours with 10 ml. Fibroblasts were obtained by treating the cells to remove connective tissue and then precipitating by centrifugation at about 700 xg for 5 minutes. The fibroblasts obtained were 5 using DMEM (manufactured by Lifetech Technologies) + 10% FCS as a medium.
The cells were subcultured and grown in a culture flask at 37 ° C. in a CO ₂ incubator while exchanging the medium every 3 days. Fibroblasts were detached from this culture flask with trypsin solution, centrifuged and collected, and DMEM + 10% FC
A cell suspension was prepared in S. 5 × 1 of this cell suspension
The inoculated atelocollagen sponge prepared in Example 1 was seeded at a cell density of 0 ⁴ cells / cm ² and DM
Cultured in EM + 10% FCS for 2 days.

At this time, before seeding the fibroblasts, FCS was evenly dropped and impregnated into the atelocollagen sponge to be impregnated and allowed to stand at 37 ° C. in a 5% CO ₂ incubator, and after 20 hours, excess FCS was removed. .

After this, the medium in the container containing the atelocollagen sponge in which the fibroblasts had been seeded and cultured was removed, and the atelocollagen sponge was turned inside out. After that, the epidermal cells cultured and proliferated in the above were added to 2 × 10 ⁵ cells.
Seed at a density of / cm ² and leave it in a clean bench for 6 hours, then add 20 ml of green medium and add 5% CO 2.
_The cells were cultured in an incubator at 37 ° C. for 14 days to obtain complex-cultured skin.

Test Example Application of Combined Cultured Skin About 2.5 × 2 cm full-thickness defect was created on the back of nude mice.

The composite cultured skin obtained in Example 2 was cut according to the shape of the defect, and a force was applied to both sides parallel to the incision to form a through hole, which was then applied. The application site was covered with a gentamicin-containing wound dressing, which was sewn together with the dressing on top of which gauze was placed and fixed with an elastic bandage.

After 3 weeks from the application, no leaching solution was visually observed, and the skin was almost entirely skinned and dried. As for the tissue, stratification of the epidermis was observed and the skin was in good condition for reconstruction.

Comparative Example Preparation of Composite Cultured Skin with No Incision The composite culture skin prepared in the same manner as in Examples 1 and 2 without the incision was applied to nude mice in the same manner as in Test Example for 3 weeks. Later macroscopic and morphological observations were made. As a result, retention of exudate was observed in a part of the applied area and reconstruction of the dermal layer was observed in the applied area, but epidermal cell engraftment was not observed.

[0065]

EFFECTS OF THE INVENTION According to the present invention, the pretreatment for the purpose of preventing the cell from falling out when the cells are seeded on the collagen substrate is not required, and the cultured skin is applied before the cultured skin is applied to the wound surface. A through hole is created by applying a force to open the notch provided in the cell to facilitate sufficient nutrient supply to seeded and cultured epidermal cells and / or fibroblasts and discharge of the exudate retained in the application site. It is possible to provide the cultured skin that can be performed at low cost.

[Brief description of drawings]

FIG. 1 is a schematic view showing a state of incision before (a) and at the time of (b) application of the cultured skin substrate or the cultured skin (incision is linear) of the present invention. The arrow in FIG. 1A indicates the direction in which a force is applied.

FIG. 2 is a schematic view showing positions where cuts are provided in the cultured skin substrate of the present invention. In (a), cuts having a length of 2 mm are provided at vertical intervals of 2 mm and at horizontal intervals of 5 mm, and in (b), cuts having a length of 2 mm are provided at vertical intervals of 2 mm.
Every 5 mm, and the horizontal intervals are 5 mm, and the cuts between adjacent rows are staggered, and (c)
In, the cuts having a length of 2 mm are provided at vertical intervals of 2 mm and horizontal intervals of 1 cm.

FIG. 3 is a schematic view showing a state of incision before (a) and at the time of (b) application of the cultured skin substrate or the cultured skin of the present invention (the incision has a Nami shape). The arrow in FIG. 3A indicates the direction in which a force is applied.

FIG. 4 is a schematic view showing a state of incision before (a) and at the time (b) of application of the cultured skin substrate or the cultured skin of the present invention (the incision has a key shape). The arrow in FIG. 4A indicates the direction in which the force is applied.

FIG. 5 is a schematic view showing the state of incision before (a) and during application (b) of the cultured skin substrate or the cultured skin (the incision is cup-shaped) of the present invention. The arrow in FIG. 5A indicates the direction in which the force is applied.

FIG. 6 shows before (a) and during (b) application of the substrate for cultured skin of the present invention or the cultured skin (radial incision is radial).
It is the schematic which shows the state of the notch. The arrow in FIG. 6A indicates the direction in which the force is applied.

FIG. 7 is a schematic view showing the state of incision before (a) and during (b) application of the cultured skin substrate or cultured skin (concentric cuts) of the present invention. The arrow in FIG. 7A indicates the direction in which the force is applied.

FIG. 8 shows before (a) and at the time of (b) application of the substrate for cultured skin of the present invention or the cultured skin (oval with concentric cuts).
It is the schematic which shows the state of the notch. The arrow in FIG. 8A indicates the direction in which the force is applied.

FIG. 9 is a schematic view showing a state of incision before (a) and at the time (b) of application of the cultured skin substrate or the cultured skin (the incision is amorphous) of the present invention. The arrow in FIG. 9A indicates the direction in which the force is applied.

Claims (20)

[Claims] 1. A substrate for cultured skin, which comprises a collagen sponge or a collagen sheet having a through cut. 2. The substrate for cultured skin according to claim 1, wherein the collagen is atelocollagen. 3. The substrate for cultured skin according to claim 1 or 2, wherein the incisions that penetrate therethrough are provided uniformly. 4. The substrate for cultured skin according to claim 1, 2 or 3, wherein the notch that penetrates is linear and is provided along one direction. 5. A piercing notch is provided at least partially in a 2.5 cm × 2.5 cm square in the cultured skin substrate, and the 1 cm × 1 cm square does not include a total incision of more than 4 cm in length. The substrate for cultured skin according to claim 1, 2, 3, or 4. 6. A cultured skin comprising a substrate for cultured skin comprising a collagen sponge or a collagen sheet provided with a through cut and a cell derived from seeded and cultured skin. 7. A cultured skin comprising a substrate for cultured skin comprising a collagen sponge or a collagen sheet having a through cut and a fibroblast derived from skin seed-cultured on at least one surface of the substrate, the culture comprising: Cultured skin capable of forming through holes by applying force to open the incision when applied to the skin. 8. A cultured skin comprising a substrate for cultured skin comprising a collagen sponge or a collagen sheet having a through cut and an epidermal cell seed-cultured on one surface of the substrate, wherein the cut is applied when the cultured skin is applied. Cultured skin that can generate through holes by applying force to open the skin. 9. A cultured skin substrate comprising a collagen sponge or a collagen sheet having a through cut, a skin-derived fibroblast cultured on at least one surface of the substrate, and seeded on one surface of the substrate. A cultured skin comprising cultured epidermal cells, which is capable of forming a through hole by applying a force to open the incision when the cultured skin is applied. 10. The cultured skin according to claim 6, 7, 8 or 9, wherein the collagen is atelocollagen. 11. The cultured skin according to claim 6, 7, 8, 9 or 10, wherein the incisions that penetrate therethrough are provided uniformly. 12. The through-hole notch is a straight line provided along one direction, and the through-hole can be generated by applying a force to open the notch.
The cultured skin according to 9, 10 or 11. 13. A piercing notch is provided at least partially in a 2.5 cm × 2.5 cm square in a cultured skin substrate, and the 1 cm × 1 cm square does not include a total incision of more than 4 cm in length. Claims 6, 7, 8,
The cultured skin according to 9, 10, 11 or 12. 14. A step of: a) providing a notch penetrating a cultured skin substrate comprising a collagen sponge or a collagen sheet, and b) a skin-derived fibroblast cell on at least one surface of the substrate with the notch closed. A method for producing cultured skin, which comprises a step of seed culture. 15. From the step of a) providing a cut-out which penetrates into a cultured skin substrate made of a collagen sponge or a collagen sheet, and b) seeding and culturing epidermal cells on one surface of the substrate with the cut being closed. Method for producing cultured skin. 16. A step of: a) providing a notch penetrating a culture skin substrate comprising a collagen sponge or a collagen sheet; and b) skin-derived fibroblasts on at least one surface of the substrate in a state where the notch is closed. A method for producing cultured skin, which comprises the steps of seed culture and seed culture of epidermal cells on one surface of the substrate. 17. The method according to claim 14, 15 or 16, wherein the collagen is atelocollagen. 18. The manufacturing method according to claim 14, 15, 16 or 17, wherein the notches that penetrate therethrough are provided uniformly. 19. The penetrating notch is a straight line provided along one direction, and the penetrating hole can be formed by applying a force to open the notch.
The production method according to 16, 17, or 18. 20. A piercing notch is provided at least partially in a 2.5 cm × 2.5 cm square in a cultured skin substrate, and the 1 cm × 1 cm square does not include a total incision greater than 4 cm in length. Claims 14, 15,
The production method according to 16, 17, 18 or 19.